Gram staining, Material and Staining Procedure
- 2. Materials required • Bacteria • Slide • Water • Dropper • Inoculating loop • Crystal violet dye • Gram’s Iodine solution • Ethyl alcohol • Safranine • Glycerin • Coverslip • Microscope
- 4. Staining of bacteria (Gram staining) • Take a slide and place a drop of water on it. • Transfer bacterial colonies from culturing plate to slide containing drop of water using inoculating loop. • Spread bacterial colonies on slide and make a thin smear. • Allow the smear to dry thoroughly.
- 5. • Heat fix the slide. • Cover the smear with crystal violet dye for 30 sec - 2 min. Rinse with water for 5 sec. • Add Grams Iodine solution (Iodine and potassium iodide), 1 min. • A decolorizer (ethyl alcohol or acetone) is added to the sample, 3 sec. • Rinse with water, 3 sec.
- 6. • A counter stain, Safranine, weakly water soluble, is added to the sample, 1 min. wash with water for 5 sec. • Apply glycerin on smear. • Place cover slip. • Observe under microscope.
- 10. Thanks
- 11. A decolorizer (ethyl alcohol or acetone) is added to the sample, 3 sec. Rinse with water, 3 sec. Complex will not penetrate the cell wall and is retained in Gram positive bacteria In Gram negative bacteria, outer membrane is degraded when treated with decolorizer and thin layer of peptidoglycan will not retain the stain and color is lost. Safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram-positive bacteria. The decolorized Gram-negative bacteria are stained red.